JPS58210021A - Preparation of cell stimulating substance - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a cell stimulant, and more particularly to a method for obtaining a solution of a cell respiration stimulating active substance from bovine blood.

The fact that cellular respiration-stimulating active substances can be obtained from cow blood is
It is described in West German Patent No. 1076888 and is well known. The method described here involves, for example, defibrinating fresh bovine blood, subjecting the resulting solution to a hemolytic reaction, and separating components of relatively high molecular weight, such as proteins in particular, from the resulting hemolyzed solution. By carrying out a dialysis operation followed by gentle concentration to a dry substance weight of 30-60 mL/ml, a solution of the active agent suitable for therapeutic use is directly obtained. The dialysis required in this method can be carried out using all known and conventional dialysis materials to achieve the objectives stated therein. In this case, the use of cellophane tubes is stated to be particularly suitable.

Although the above-described method provides products with the desired therapeutic activity, this method has the disadvantage that the necessary dialysis is relatively time-consuming and difficult to operate. Moreover, the obtained products comply with the upper limit of molecular weight.

It is not the same homogeneous product because its composition is variable. Furthermore, the yield of the product obtained is also not satisfactory.

Furthermore, the process of DE 1076888 gives products with a relatively high content of inorganic salts, in particular sodium chloride and potassium chloride. Such salts cannot be separated and removed by the method described therein. The salt content of the active substance solution obtained is, for example, approximately 25% of the solids content.
~80% by weight, making corresponding injection solutions or highly concentrated topical formulations several times hypertonic. Due to their hypertonic nature, intramuscular administration of such active substance solutions is painful and cellularly damaging;
This is detrimental to the desired cell regeneration effect.

This undesirably excessive salt content of the cellular respiration stimulant obtained by the process of DE 1076888 is particularly important in order to obtain an isotonic or only slightly hypertonic active agent solution.
It could in principle be lowered by various methods, such as dialysis, ultrafiltration, ion exchange or gel filtration. However, all these methods are either completely useless or, if they are useful, give very unsatisfactory results. According to DE 2512936, the desalination or partial desalination of the cellular respiration stimulating active substance solutions in question can be carried out by a very special gel filtration method, i.e. this cellular respiration stimulating active substance solution containing a high concentration of salts. The active substance solution is passed through a filtration layer made of a highly crosslinked gel placed on a perforated centrifugal drum at a solution to gel volume ratio of 1:2.5 to 1.4.
It is said that this can be achieved by contacting them at a ratio of 5. This West German patent also details the drawbacks of other known processes, which in principle allow partial desalination. All possible methods, including the method described in this West German patent no. The disadvantage is that it requires the use of equipment.

Furthermore, simultaneous removal of the desired inorganic salts results in an adsorption of the cellular respiration-stimulating active agent onto the separation medium, so that more or less active substance is lost. The regeneration of the gel and ion exchange resin, which is absolutely necessary when carrying out these methods, is very time consuming and only batchwise operation is possible, and furthermore the separation conditions have to be constantly changed.

The method for preparing cellular respiration-stimulating active substances described in West German Patent No. 1076888 has various drawbacks for the reasons mentioned above.
These disadvantages could not be overcome even by the procedure described in German Patent No. 2,512,936 for separating the excessively high content of undesirable inorganic salts.

It is therefore an object of the present invention to solve the problems of known methods of operation.
In particular, a process for obtaining natural cellular respiration-stimulating active substances which can be carried out economically and continuously and which can be completely adjusted to obtain products with a constant upper and lower molecular weight limit. The object of the present invention is to provide a novel method in which a solution of a cell respiration stimulating active agent can be directly obtained by a controlled partial desalination operation.

The method of the present invention for obtaining a solution of a cell-stimulating active substance from bovine blood consists of removing cellulose from fresh bovine blood (defibrinization), hemolyzing the defibrinated live blood, and dissolving the hemolysed bovine blood. is subjected to membrane ultrafiltration using an ultrafiltration membrane with a molecular weight exclusion limit of about 8,000 Daltons or more, and the ultrafiltrated liquid containing a respiratory stimulant active substance with a molecular weight of about 8,000 Daltons or less is collected, The collected ultrafiltrate is subjected to electrodialysis using an electrodialysis membrane with a permeability up to about 300 daltons to partially remove inorganic salts and other ionic low molecular weight substances from the cell respiration stimulating active substance solution. It consists of a process of separation.

By the method of the present invention, therapeutic substances which have basically the same cellular respiration stimulating activity as by the method of West German Patent No. 1076888, but with various significant improvements due to the combined use of ultrafiltration and electrodialysis, can be obtained. is obtained. Ultrafiltration provides a homogeneous product with clear separation of proteins and other high molecular weight substances, while at the same time reducing the time required.
There is also an economical improvement in terms of space requirements and yield.

The fact that the solution obtained by ultrafiltration can be used for electrodialysis after reducing the container size to some extent means that
On the one hand, relatively highly concentrated solutions, e.g. solids content of 1
It has the advantage that solutions which are between 5 and 30% by weight can be used and thus can be treated in a virtually constant equilibrium after the initial phase with an ion exchange membrane. By varying parameters such as current density, temperature and time, the desalination operation can be controlled in a certain direction and adjusted to minimize the loss of organic matter.

The individual degree of desalination can be determined very easily by continuous measurement of electrical conductivity or continuous measurement of osmotic pressure.

The known or inventive operations in the process of the invention, unless otherwise specified, are carried out as follows: The bovine blood used as starting material is stirred vigorously and, if desired, immediately after collection. cooling), the generated cellulose (
Fibrin) is filtered and defibrinated. This operation
This is usually done directly at the slaughterhouse.

After defibrinated raw blood, the blood, if desired, is mixed with a preservative and immediately frozen and stored under frozen conditions until use.

To liberate cellular respiration-stimulating active substances, the defibrinated blood or blood frozen for storage must be hemolyzed, ie the red blood cells must be disrupted). For this purpose, it is necessary to destroy the cell membranes of blood cells in some way.

Such destruction can be carried out both chemically and mechanically. To chemically lyse the blood, for example, defibrinated blood is treated with enzymes or bacteria that attack and destroy cell membranes. Mechanical solubility is a substance that increases intracellular osmotic pressure,
This can be achieved, for example, by adding water or an organic solvent such as ethanol, diethyl ether or acetone, thereby disrupting the cell membrane.

Alternatively, the blood can be lysed by gradual cooling, so-called ice-hemolysis, so that relatively large ice crystals can form that penetrate the cell membrane. After hemolysis, virtually all the red blood cells are destroyed and their contents are liberated.

The hemolytic reaction can also be carried out in the presence of a preservative.

The blood thus obtained is subjected to the first step of the method of the invention, namely an ultrafiltration operation using a membrane with an exclusion limit of molecular weight of about 8000 Daltons, preferably about 5000 Daltons or more. can.

Membranes used for ultrafiltration can be of a variety of materials, including:
Triacetylcellulose or hydrophilic polyolefin membranes are preferred.

A membrane of the above nature with an exclusion limit of molecular weight of 8000 daltons or more can contain 1 molecule of dextran of 2oooo.
It is characterized by its permeability to separate molecules (dextran with a molecular weight of 810,000 with a nominal separation of 85%, polyethylene glycol with a molecular weight of 6,000 with a nominal separation of 50%, and lactose with a molecular weight of 342 with a nominal separation of 1 OeI).

A membrane with an exclusion limit of about 5,000 daltons or more has a permeability of 100% separation for dextran of molecule tloo00, 97% separation of polyethylene glycol of molecule it 6000, and 30% separation of polyethylene glycol of molecular weight 1000. Characterized by separation.

After defibrillation and hemolysis, the solution obtained by ultrafiltration using a membrane with the above properties has an upper limit of molecular weight of about 8000.
Dalton or approximately 5000 Dalton finished product.

In order to carry out continuous multi-step ultrafiltration, it is convenient to use an ultrafiltration apparatus consisting of four units as shown in FIG. 1 and detailed in Example 1.

This device contains an ultrafiltration module called Model 131, which is 1) aterson
Candy International Ltd
Reverse Osmosisr)ivisio
n, Whitchurch, Great Writ
ain in the brochure BPL 3/73 2M and in West German Patent Application No. 2065812.

This particular device uses a membrane with a molecular weight exclusion limit of about 8000 Daltons or greater. However, the device can be similarly fitted with membranes having different expulsion limits, such as molecular weight expulsion limits of about 5000 Daltons or more.

This ultrafiltration device uses a pressure of e.g. 8 to 10 bar and filters the solution to be ultrafiltered e.g.
Cool and operate. The device contains conventional preservatives such as 4-hydroxybenzoic acid methyl ester or 4-hydroxybenzoic acid methyl ester.
A bovine blood solution that has been preserved in an alcoholic solution of hydroxybenzoic acid propyl ester (preferably an ethanolic solution of a mixture of these two preservatives) can be added.

During the first and final steps of multi-5tep ultrafiltration, the drained ultrafiltrate is used as a diluent for the concentrated and re-ultrafiltrated blood solution. Add an amount of preservative solution to compensate for the
Preferably, the level of the blood solution to be ultrafiltered again in the next step is kept approximately constant. Preferably, all steps but the last step of this continuous multi-step ultrafiltration process are carried out while the level of the blood solution to be ultrafiltered is maintained approximately constant. Only in the final step do not dilute the blood solution from the penultimate step, allowing this volume reduction to dialyze all the blood load.

Finally, waste blood generated from dialysis is discarded.

The filtrate obtained by ultrafiltration, which is free of proteins and relatively high molecular weight substances, is subjected to a volume reduction step at a temperature not exceeding 40° C. under reduced pressure. By distilling this solution under reduced pressure at a temperature of, for example, 30 to 35°C,
Its capacity is determined by the specific gravity of 110 to 1.15 at 20°C.
g-/ml. The purpose of this concentration is, in part, to remove the organic solvent used for hemolysis, or which was needed for a particular preservative, together with the preservative; In 2,
This is to concentrate the ultrap-permeate solution to a dry content suitable for carrying out the next electrodialysis step of the process of the invention, for example a dry content of 180-230'rK//ml.

Preservatives precipitated by this concentration are best separated by filtration, but any preservatives that may remain in solution can be removed by adjusting the pH value of the solution, e.g.
-(5) can be precipitated by adding concentrated hydrochloric acid, which is then filtered. The distillation necessary for this concentration is conveniently carried out using a suitable vacuum evaporator.

The concentrate obtained in the above concentration is subjected to the second step of the present invention, that is,
The inorganic salts contained therein are partially separated and removed,
Osmotic pressure of 250-550 milliosmoles (mo S mo 1), conductivity of 40-75 mS/c+n or 20
Specific gravity at ℃ is 1.05-1.08! molecular weight of about 3 to obtain an isotonic to slightly hypertonic solution of i'/ml.
Cell stacks consisting of alternating cation- and anion-exchange membranes with membrane permeability up to 0.00 Daltons, preferably a molecular weight of about 400 Daltons.
) to an electrodialysis process.

Membranes used for this purpose are characterized by the permeability of inorganic or organic ions with a molecular weight of up to about 300, or preferably up to about 400, but not of oligopeptides and relatively large molecules. It will be done.

Using such a membrane, the active substance has a
A lower limit for molecular weight permeability of Daltons, or preferably about 400 Daltons, is given.

Electrodialysis can perform this step of the invention, for example, from 5 to 70 m
A/-free membrane cross section, preferably 20 to 50 mkl-current density of free membrane cross section, 0.2 to 0.2 per membrane pair (membrane Pa1r)
2V.

It can be carried out under conditions customary for the separation of inorganic salts from corresponding aqueous concentrates, such as preferably at a DC voltage of 0.5 to IV per membrane pair and a temperature range of 5 to 30°C. .

The electrodialysis process described above has a specific desired permeability.
This can be carried out using a conventional electrodialysis apparatus of cell stacks with alternating cation- and anion-exchange membranes. For example, an electrodialysis device shown in FIG. 2 and described in detail later is used.

This is Ber gho f GmbH1T↓i3b
ingen, a West German device called the BEL2 type. The membrane of this device has a membrane permeability up to a molecular weight of about 400 Daltons. A single stack of this electrodialysis device is described in detail in German Patent Application No. 2946284.

Depending on the desired mode of administration, the aqueous solutions containing cellular respiration-stimulating active substances obtained by the method of the invention can also be used directly for treatment and, if desired, can be further diluted with pyrodiene-free water. Alternatively, it may be further concentrated if necessary, so that the corresponding pharmaceutical formulations can be, for example, solutions, aerosols, pastes, creams or gels. This cell respiration stimulating activity is essentially the same as that of the substance obtained in West German Patent No. 1076888, as already mentioned. Such pharmaceutical preparations are therefore used in particular for promoting and controlling the therapeutic process. The dosage of active substance used for this purpose will vary depending on the method of administration and the nature and severity of the condition to be treated.

Injuries which can be treated with the cellular respiration stimulating active substances obtained by the method of the invention are, for example, burns, ulcers, ganglia and the like.

For this purpose, the active substance can be administered intravenously, intraperitoneally, intramuscularly or, in the case of an open side, locally. In the case of injection therapy, such as in the treatment of ulcers in humans, 200 to 200 mm of the active substance per day is administered intravenously or intraarterially, and then as a follow-up treatment, 80 to 200 ``I!?'' of the active substance per day is administered intravenously. Administer intravenously or intramuscularly.

This and other uses of this active substance include:
sfelden, Swiss National Brochure' 5ol
coseryl reaktiviertden
gesLorten Energies toffwe
chsel der Zelle @ (Publication number: 5s
-cr-r/d-6, sl).

It is important for the successful implementation of the method of the invention that the biological activity of the bovine blood extract is unaffected, especially during ultrafiltration, and more especially during electrodialysis. When carrying out the method, the original biological activity must be fully preserved. According to the method of the invention, a blood extract is obtained which corresponds at least to the biological activity of the product obtained according to West German Patent No. 1,076,888 or even more.

The risk of possible loss of biological activity in the ultrafiltration step carried out according to the method of the invention is considered to be low:
This is because, in this step, only proteins and substances of relatively high molecular weight are separated, and most of the biological activity measured by corresponding experiments is due to the relatively small amount of cellular respiration-stimulating active substances that can be recovered by the method of the present invention. This is because it is in a low molecular weight range. It is also conceivable that ultrafiltration does not affect the blood extract any more than does simple dialysis carried out according to the method of DE 1076888. Comparative tests of the possible effects on biological activity of the ultrafiltration of the present invention are therefore not necessary.

Similarly, the concentration of the solution following ultrafiltration is also carried out under conditions that do not have any effect on the biological activity of this blood extract. Therefore, no comparative tests are necessary for this step either.

On the other hand, separation of inorganic salts contained in active substance concentrates by electrodialysis may have a negative effect on the biological activity of blood extracts. A relatively large amount of salts has to be separated here, and the molecular weight of the salts to be separated is relatively close to the lower limit of the molecular weight of the cellular respiration-stimulating active substance obtained by the method of the invention. It is therefore necessary to investigate in a suitable manner whether the biological activity of the active substance solution obtained by electrodialysis is affected compared to the starting material concentrate. It is also necessary to investigate how the physical properties are affected by electrodialysis compared to the starting material concentrate, and what important substances other than inorganic salts are removed. Of course it doesn't.

Individual bioactivity level comparison tests are carried out in three ways: a) carbonate with
b) testing of 02 metabolism enhancing activity by Warburg (giving Warburg activity); and C) standardized, Test for wound healing promotion activity in back burns (
(gives the healing time of the wound when contrasted with saline).

The first two in vitro tests showed that full biological activity was retained under all test conditions and that it was rather partially improved since cell-damaging salts were no longer present. . Wound healing tests showed the expected results that the partially desalinated blood extract obtained by the method of the invention was well tolerated.

In all these comparisons, the activities of the non-desalted starting concentrate and the partially desalted extract are of course under standardized conditions, i.e. solutions of the same organic active substance content, but not the same dry content. Do it using For this reason, the solutions to be compared are always diluted to a constant total nitrogen content, for example i 1 m9 nitrogen per ml of injection solution.

This total nitrogen content is believed to represent the content of organic substances in each solution. Of course, solutions used for therapeutic purposes can also be tested in exactly the same way;
The content is also determined by the Warburg test and the fibroblast test.

Changes in dry weight, conductivity, osmotic pressure and ash content are investigated by comparative tests. Differences (changes) between the starting material concentrate and that obtained by separating and removing low molecular weight substances, i.e. mainly anions and other low molecular weight substances, are compared by chemical analysis for parameters and characteristic substances. Find out by

The results of comparative experiments carried out on two starting concentrates and eight partially desalted extracts obtained by the process of the invention are shown in Table 1 of the Examples. From this table, it can be seen that in addition to inorganic salts, lower carbonic acids and amino acids have been removed, but the biological activity is completely retained.

In order to show that electrodialysis carried out according to the method of the invention does not significantly change, or only slightly, the composition of the blood extract with respect to its organic components, high-pressure liquid chromatography I did this. The chromatograms of the starting concentrate and partially desalted extract obtained were always the same.

The present invention will be explained in more detail with reference to Examples below.

Example Ultrafiltration Ultrafiltration apparatus of the type described above (Paterson Ca
ndy International Ltd, (7)
B1) is used. This four-stage ultrafiltration device and its function can be understood from FIG.

Each step (steps 1 to 4) of the total 4-step equipment requires 1.7M
Two ultrafiltration tube modules (U-U4) with filtration surfaces are provided. The membrane is comprised of acetylcellulose having the permeability properties previously described, ie, a molecular weight permeation limit of about 8000 Daltons or more. The pressure transmission pumps P1 to P4 have a pressure of about 1oOo /
It is a rotary piston pump with a capacity of /h. Supply pumps P5-P7 and discharge pump P8
~P1o is a dosage pump that can be freely adjusted in the range of 2 to 201/h. 4 tanks T0
~T4 all contain approximately 300 min each. The added solution was placed in a cooler cooled by cooling water for 4 to 18 hours.
The temperature is maintained at ℃. All PIs represent pressure display means, and all Fl represent flow rate display means. Other abbreviations have the following meanings: ] 3 - Defibrillated and dissolved blood solution KL - Preservative solution UF - Ultrafiltrate Al Step 1), T
(Step 2) and T3 (Step 3), 4-
Defibrillation containing 15% by volume of a 2% (weight/volume) alcohol solution of a 9:IC weight/weight) mixture of hydroxybenzoic acid methyl ester and 4-hydroxybenzoic acid propyl ester as a preservative. 250 doses of hemolyzed bovine blood B were supplied. Next, when the pressure transmission pumps P□ to P3 are activated, the ultrafiltrate UF begins to flow out, and the contents of the tanks T to T3 decrease at the same rate. As soon as the content of the tanks Tl-T3 reaches approximately 150 nos, feed pumps P5-P7 and discharge pumps P8-P1o are activated. 101/h already mentioned,
Defibrinated and hemolysed bovine blood containing preservative is supplied by pump P5. At the same time, 9=1 of 4-hydroxybenzoic acid methyl ester and 4-hydroxybenzoic acid propyl ester
(w/w) 1% (wt/vol) of the mixture 101/h of preservative solution KI- of 15 vol.% alcohol solution and 85 vol. % water is added to tanks T2 and T3, respectively, by feed pumps P6 and PI. supply This dilutes the ultrafiltered packing (I oad ). Adjust the discharge pumps P8-P1o so that the level in the tanks T1-T3 remains constant at 150 rpm.

Thereby, each of the tanks T2 and T3 is supplemented with the amount of ultrafiltrate UF flowing out from the individual ultrafiltration tube modules U2 and U3, and a diluent is added so that a constant level of blood filling is maintained. Supply KL. Tank T
The technique also keeps the blood filling at the same level. Only in tank T4 is the blood fill level reduced during the performance of this continuous multi-step ultrafiltration process by the amount of ultrafiltrate exiting ultrafiltration tube module U4. This is because no diluent is added here to keep the blood fill level at the same level as in the previous step.

Immediately after approximately 120 tons of solution is supplied from step 3 to tank T4 (step 4), pressure transmission pump P4 is switched on. The waste blood discharged from the flow channel of this tank T4 is discarded.

All four steps reach equilibrium after about 36 hours.

The ultrafiltrate UF flowing out from the ultrafiltration tube modules U1 to U4 is typical of this operation, and is collected and used in the next concentration step. The flow rate of the ultrafiltrate LJ l" is approximately 4.7 n/b in step 1 and approximately 11 n/b in step 2.
/h, and in step 3, about 10.4 /h.

In step 4, it is about 18 rpm. Therefore, the amount of ultrafiltrate transferred is 5.3 l/h in the discharge pump P8, 4.3 n/h in the discharge pump P9, and 3.9 n/h in the discharge pump P1o, while the amount in the tank T4 is The outflow of waste blood AB from (step 4) is 2. It is IJ2/h.

Concentration Ultrafiltrate UF obtained by the above four-stage ultrafiltration
was distilled at a temperature of 35°C under a reduced pressure of 30 mbar to give a specific gravity of 1.130 m'i at 20°C.
/ml, thereby removing the alcohol contained in the preservative solution.

This concentration precipitates most of the preservative, which is then washed away. When the obtained P solution is treated with a small amount of concentrated hydrochloric acid until the pH reaches 5, the remaining preservative will precipitate, and this will be removed again.

The material obtained by the above ultrafiltration and concentration is collected and used as starting material (i.sigma.7) for the next step of the method of the present invention, electrodialysis.
33.10).

Repeat ultrafiltration and concentration Using bovine blood of different origin, the above two steps are repeated to produce another starting concentrate C! 350.03) and subjected to the next step of electrodialysis.

The starting concentration obtained on electrodialysis means 733.10 and! 350.0
3, the former into two parts and the latter into six parts,
Each is separately applied to the electrodialysis apparatus shown in FIG.

In this way, starting concentrate! 733. Electrodialysates AI 2 and A13 using IO starting concentrate, 1350
．． 03 to obtain electrodialysate layers 14, A15, 16, 17, A18 and A19.

Electrodialysis equipment of the type already described (Berghof Gm
BF from 1tI, L2) is used. This device and its functions are schematically shown in FIG.

The central part of the device is the cell stack, which consists of seven pairs, each consisting of an anion exchange membrane a and a cation exchange membrane K (types CMV and AMV), with one membrane on each side as the last membrane. K (however, only three pairs of ion exchange membranes are shown in the diagram for simplicity). The membrane used has a permeability of up to about 400 molecules. The following three liquid circuits are connected to this cell stack: The solution to be desalinated is circulated from tank T to pump P0 and cooler via □. This is called dilute D, as in the literature.

The aqueous solution that picks up the salts and increases their concentration is circulated from tank T2 via pump P2 and cooler. This solution is called a concentrate.

A 2% (weight/volume) sodium sulfate aqueous solution is circulated from tank T3 via pump P3 and is referred to as electrode cleaning solution EI-.

The heat generated by friction is removed by coolers 1 and 2, thus keeping the solution at 25°C.

The conductivity measuring cell LF is placed in the tileate circuit after the cooler. The flow rate of the concentrated descending ultrafiltrate UK is regulated by a two-point regulator k.

A part of the concentrated descending salt solution KA is continuously carried away on the concentration side, and the pump P5 replenishes that amount with distilled water W. Furthermore, water moves from the dilution side to the concentration side within the cell stack.

After having its conductivity measured in a conductivity measuring cell, a part of the tile knee) D is continuously pulled out and passed through a sterile filter SF.
After passing through, it enters tank T4 as finished product PR, where it is stored at 0° C. until it is processed into the corresponding pharmaceutical formulation.

To start the operation of the electrodialysis apparatus, the concentrated ultrafiltrate UK1,27 obtained in the above-described manner is charged into a 21-capacity tank. Pour 1 liter of distilled water into tank T2 of the same size,
Add 750 rnl of 2% N 404 solution to tank T3. After filling the cell bundle and the entire circuit and removing the air, the pump P0 ~ transports approximately 90 z/h, respectively.
Activate P3. Direct current is connected to two electrode plates (+ and -)
Pass it through. In this case, the voltage is selected so that a current of about 20 mA/- flows through the cross section of the free membrane. Therefore, the free membrane cross section is 3
A total current of approximately 75 mA flows through the cell stack No. 7-. The starting voltage of about 16V is based on a cell structure consisting of 7 pairs of membranes!
Adjust to lower the normal value to about 5.5■ within 1 hour.

The conductivity dropped to 45-50 mS/cm after 10 hours. The feed rate of the concentrated ultrafiltrate UK is adjusted so that the conductivity of the tileate is on average about 100, l/h, 45~
Adjust the feed pump P4 to the regulator so that it is 50m5/cm.
Adjust through.

Feed pump P5 is set at 200 ml/11 and the discharge of salt concentrate KA from tank T2 is approximately 220 ml/11.
”.

Two starting concentrates IG, 733.10 and 5350.
The results of the above dialysis repeated several times using 03, and various electrodialyzers 12, 13, 141. [16,)
The compositions of A17, K, l8 and y(G, 19) are shown in Table 1.

Table 1 also lists the temperature, current density and DC voltage conditions employed, analytical data of the starting concentrate and recovered electrodialysate, and comparative data of biological specific activity.

The concentrated tile ate obtained by the above electrodialysis is diluted with sterile water, for example, 5 times, and the
It is convenient to prepare isotonic to slightly hypertonic active substance solutions with an osmotic pressure in the milliosmolar range.

Tests have shown that the method according to the invention produces solutions of cellular respiration-stimulating active substances which have at least the same or partly better biological activity than the corresponding active substance solutions obtained by known methods, or from bovine rrn fluid. Especially found out what you can get in a smart and economical way.

A value of 1.0 or higher is valid: Quantitative low concentration is impossible data based on

[Brief explanation of drawings]

Figure 1 is a schematic diagram of the ultrafiltration process, and Figure 2 is a schematic diagram of the electrodialysis process. U□～U4・・Ultrafiltration device, K・・
-Cation exchange membrane, a... anion exchange membrane.

Claims (1)

[Scope of Claims] 1. Bovine blood immediately after blood collection is vigorously stirred, the generated cellulose is filtered and defibrillated, the resulting solution is hemolyzed, and about 8,000 ml of blood is contained in the hemolyzed solution. Substances and proteins having a molecular weight of more than Dalton are separated by membrane separation method, and this solution from which proteins and high molecular weight substances have been removed is heated under reduced pressure at a temperature not exceeding 40°C until the density at 20°C is 1.
The inorganic salts are partially separated from the resulting concentrate, and the resulting concentrate is finally diluted as desired to give an osmotic pressure of 250 to 550 milliosmol. A method for the preparation of cellular respiration-stimulating active substances with a molecular path in the range of about 300 to 8000 Daltons, comprising obtaining an isotonic to slightly hypertonic solution with a molecular weight of about 8000 Daltons. It is carried out using a continuous multi-step ultrafiltration method using a membrane with the above molecular weight exclusion limit,
A method characterized in that the partial separation of the inorganic salts is carried out by electrodialysis using a cell stack having alternating cation and anion exchange membranes having membrane permeability up to a molecular weight of about 300 daltons. 2. In ultrafiltration, use a membrane with a molecular weight exclusion limit of about 5,000 Daltons or more, and in electrodialysis, use a membrane with permeability up to about 400 Daltons, in the range of about 400 to 5,000 Daltons. 2. The method according to item 1 for preparing a cell respiration stimulating active substance having a molecular weight of . 3.5-70mA/-free membrane cross section, preferably 20-5
01 nA/crrl current density across the free membrane cross section, a DC voltage of 0.2 to 2 cm per membrane, preferably 0.5 to 1 cm, and a temperature range of 0 to 30 °C. The method described in section. 4. In each step between the first step and the final step of continuous multi-step ultrafiltration, adding a diluent in an amount to compensate for the amount of ultrafiltrate flowing out from each ultrafiltration module, 4. The method according to any one of items 1 to 3, wherein the blood solution to be re-ultrafiltered in the next step is maintained at a substantially constant level. 5. 5. The method according to any one of items 1 to 4, wherein the blood solution to be ultrafiltered is maintained at a substantially constant level in all steps except the final step of continuous multi-step ultrafiltration.